Transgenic Research

, Volume 18, Issue 2, pp 163–171

Production of Prnp−/− goats by gene targeting in adult fibroblasts

  • Caihong Zhu
  • Bei Li
  • Guohua Yu
  • Jianquan Chen
  • Huiqing Yu
  • Juan Chen
  • Xujun Xu
  • Youbing Wu
  • Aimin Zhang
  • Guoxiang Cheng
Original Paper

DOI: 10.1007/s11248-008-9220-5

Cite this article as:
Zhu, C., Li, B., Yu, G. et al. Transgenic Res (2009) 18: 163. doi:10.1007/s11248-008-9220-5

Abstract

Homozygous mice devoid of functional Prnp are resistant to scrapie and prion propagation, but heterozygous mice for Prnp disruption still suffer from prion disease and prion deposition. We have previously generated heterozygous cloned goats with one allele of Prnp functional disruption. To obtain goats with both alleles of Prnp be disrupted which would be resistant to scrapie completely, a second-round gene targeting was applied to disrupt the wild type allele of Prnp in the heterozygous goats. By second-round gene targeting, we successfully disrupted the wild type allele of Prnp in primary Prnp+/− goat skin fibroblasts and obtained a Prnp−/− cell line without Prnp expression. This is the first report on successful targeting modification in primary adult somatic cells of animals. These cells were used as nuclear donors for somatic cell cloning to produce Prnp−/− goats. A total of 57 morulae or blastocytes developed from the reconstructed embryos were transferred to 31 recipients, which produced 7 pregnancies at day 35. At 73 days of gestation, we obtained one cloned fetus with Prnp−/− genotype. Our research not only indicated that multiple genetic modifications could be accomplished by multi-round gene targeting in primary somatic cells, but also provided strong evidence that gene targeting in adult cells other than fetal cells could be applied to introduce precise genetic modifications in animals without destroying the embryos.

Keywords

Prion disease Prnp Second-round gene targeting Adult fibroblasts 

Abbreviations

DMSO

Dimethyl sulfoxide

Dpl

Doppel protein

FBS

Fetal bovine serum

GMEM

Glasgow minimal essential medium

neo

Neomycin phosphotransferase gene

Prnp

Prion protein gene

PrP

Prion protein

puro

Puromycin N-acetyl-transferase gene

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Caihong Zhu
    • 1
  • Bei Li
    • 1
    • 2
  • Guohua Yu
    • 2
    • 3
  • Jianquan Chen
    • 2
    • 3
  • Huiqing Yu
    • 2
    • 3
  • Juan Chen
    • 2
    • 3
  • Xujun Xu
    • 2
    • 3
  • Youbing Wu
    • 2
    • 3
  • Aimin Zhang
    • 1
    • 2
  • Guoxiang Cheng
    • 1
    • 2
    • 3
  1. 1.School of Life Science and TechnologyTongji UniversityShanghaiChina
  2. 2.Shanghai Transgenic Research CenterShanghaiChina
  3. 3.Shanghai Genon Bio-Engineering Co. LtdShanghaiChina

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